Allogeneic hematopoietic stem ceil transplantation remains the only curative option for many patients with hematological malignancies. Graft-versus-host disease (GvHD) is a major limitation to transplantation of hematopoeitic cells across histocompatibility barriers, and effectively limits transplantation to a minority of patients who would benefit from treatment. Extensive T cell depletion of the donor graft can eliminate GvHD. A significant consequence of T cell depletion, however, is a profound and long-lasting T cell immunodeficiency state post-transplant resulting in severe opportunistic viral and fungal infections that significantly limit survival. The adoptive infusion of mature, memory T cells post-transplant may offer protection against opportunistic infection, and shorted the period of immunodeficiency until successful immune reconstitution occurs. The success of this strategy in reducing mortality following T cell depleted transplants, however, will depend on the selective removal of alloreactive T cells that mediate GvHD, while retaining a high repertoire of memory T cells capable of reacting to viral and third party antigens. Although a number of methods have been investigated, the extent of depleting alloreactive T cell has been limited only to <3 log, which has not fully prevented GvHD and limited the number of T cells that can be infused to improve immunity. Either incomplete in-vitro activation of donor T cells with alloreactive potential and/or inefficient depletion of the activated cells likely contributes to suboptimal depletion of alloreactivity. The overall goal of this proposal is to develop a system for efficient depletion of alloreactive T cells for clinical use. Specifically, we aim to 1) Optimize the conditions for activating the maximum number of donor-specific T cells by studying culture conditions and the kinetics of different antigens expressed selectively on activated T cells for use as targets in depletion, and 2) Develop a GMP grade hiqh-performance immunomagnetic separation system, quadrupole magnetic cells sorting (QMS), for clinical scale depletion of activated donor T cells that is capable of >=3 log depletion of alloreactive T cells while retaininq >80% of third party reactivity. The technology developed in this proposal should greatly facilitate the development of clinical trials of adoptive donor T cell therapy to improve immune function following T-cell-depleted mismatched stem cell transplants.